Ulrika Widegren

Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle

The molecular signaling mechanisms by which muscle contractions lead to changes in glucose metabolism and gene expression remain largely undefined. We assessed whether exercise activates MAP kinase proteins (ERK1/2, SEK1, and p38 MAP kinase) as well as Akt and PYK2 in skeletal muscle from healthy volunteers obtained during and after one‐leg cycle ergometry at ∼70% VO2max. Exercise led to a marked increase in ERK1/2 phosphorylation, which rapidly decreased to resting levels upon recovery. Exercise increased phosphorylation of SEK1 and p38 MAP kinase to a lesser extent than ERK1/2. In contrast to ERK1/2, p38 MAP kinase phosphorylation was increased in nonexercised muscle upon cessation of exercise. Phosphorylation of the transcription factor CREB was increased in nonexercised muscle upon cessation of exercise. Exercise did not activate Akt or increase tyrosine phosphorylation of PYK2. Thus, exercise has divergent effects on parallel MAP kinase pathways, of which only p38 demonstrated a systemic response. However, our data do not support a role of Akt or PYK2 in exercise/contraction‐induced signaling in human skeletal. Activation of the different MAP kinase pathways byphysical exercise appears to be important in the regulation of transcriptional events in skeletal muscle.—Widegren, U., Jiang, X.‐J., Krook, A., Chibalin, A. V., Bjo¨rnholm, M., Tally, M., Roth, R. A., Henriksson, J., Wallberg‐ Henriksson, H., Zierath, J. R. Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle. FASEB J. 12, 1379– 1389 (1998)

Effects of concentric and eccentric contractions on phosphorylation of MAPKerk1/2 and MAPKp38 in isolated rat skeletal muscle

1 Exercise and contractions of isolated skeletal muscle induce phosphorylation of mitogen‐activated protein kinases (MAPKs) by undefined mechanisms. The aim of the present study was to determine exercise‐related triggering factors for the increased phosphorylation of MAPKs in isolated rat extensor digitorum longus (EDL) muscle. 2 Concentric or eccentric contractions, or mild or severe passive stretches were used to discriminate between effects of metabolic/ionic and mechanical alterations on phosphorylation of two MAPKs: extracellular signal‐regulated kinase 1 and 2 (MAPKerk1/2) and stress‐activated protein kinase p38 (MAPKp38). 3 Concentric contractions induced a 5‐fold increase in MAPKerk1/2 phosphorylation. Application of the antioxidants N‐acetylcysteine (20 mM) or dithiothreitol (5 mM) suppressed concentric contraction‐induced increase in MAPKerk1/2 phosphorylation. Mild passive stretches of the muscle increased MAPKerk1/2 phosphorylation by 1.8‐fold, whereas the combination of acidosis and passive stretches resulted in a 2.8‐fold increase. Neither concentric contractions, nor mild stretches nor acidosis significantly affected phosphorylation of MAPKp38. 4 High force applied upon muscle by means of either eccentric contractions or severe passive stretches resulted in 5.7‐ and 9.5‐fold increases of phosphorylated MAPKerk1/2, respectively, whereas phosphorylation of MAPKp38 increased by 7.6‐ and 1.9‐fold (not significant), respectively. 5 We conclude that in isolated rat skeletal muscle an increase in phosphorylation of both MAPKerk1/2 and MAPKp38 is induced by mechanical alterations, whereas contraction‐related metabolic/ionic changes (reactive oxygen species and acidosis) cause increased phosphorylation of MAPKerk1/2 only. Thus, contraction‐induced phosphorylation can be explained by the combined action of increased production of reactive oxygen species, acidification and mechanical perturbations for MAPKerk1/2 and by high mechanical stress for MAPKp38.

Adiponectin protects against myocardial ischaemia-reperfusion injury via AMP-activated protein kinase, Akt, and nitric oxide

AIMS Cardiovascular disease and type 2 diabetes mellitus are associated with low plasma concentration of adiponectin. The aim of this study was to investigate whether adiponectin exerts cardioprotective effects during myocardial ischaemia-reperfusion and whether this effect is related to the production of nitric oxide (NO). METHODS AND RESULTS Isolated rat hearts were subjected to 30 min of either global or local ischaemia followed by 60 min of reperfusion. The hearts received vehicle, adiponectin (3 microg/mL), the NO-synthase inhibitor nitro-l-arginine (L-NNA) (0.1 mM), or a combination of adiponectin and L-NNA at the onset of ischaemia. Haemodynamics, infarct size, and expression of endothelial NO-synthase (eNOS), AMP-activated protein kinase (AMPK), and Akt were determined. Adiponectin significantly increased left ventricular function and coronary flow during reperfusion in comparison with the vehicle group. Co-administration of L-NNA abrogated the improvement in myocardial function induced by adiponectin. Infarct size following local ischaemia-reperfusion was 40 +/- 6% of the area at risk in the vehicle group. Adiponectin reduced infarct size to 19 +/- 2% (P < 0.01). L-NNA did not affect infarct size per se but abolished the protective effect of adiponectin (infarct size 40 +/- 5%). Phosphorylation of eNOS Ser1177, AMPK Thr172, and Akt Ser 473 was increased in the adiponectin group (P < 0.05). CONCLUSION Adiponectin protects from myocardial contractile dysfunction and limits infarct size following ischaemia and reperfusion by a mechanism involving activation of AMPK and production of NO.

Exercise-induced mitogen-activated protein kinase signalling in skeletal muscle.

Exercise training improves glucose homeostasis through enhanced insulin sensitivity in skeletal muscle. Muscle contraction through physical exercise is a physiological stimulus that elicits multiple biochemical and biophysical responses and therefore requires an appropriate control network. Mitogen-activated protein kinase (MAPK) signalling pathways constitute a network of phosphorylation cascades that link cellular stress to changes in transcriptional activity. MAPK cascades are divided into four major subfamilies, including extracellular signal-regulated kinases 1 and 2, p38 MAPK, c-Jun NH2-terminal kinase and extracellular signal-regulated kinase 5. The present review will present the current understanding of parallel MAPK signalling in human skeletal muscle in response to exercise and muscle contraction, with an emphasis on identifying potential signalling mechanisms responsible for changes in gene expression.

Effect of exercise and training on phospholemman phosphorylation in human skeletal muscle.

Phospholemman (PLM, FXYD1) is a partner protein and regulator of the Na(+)-K(+)-ATPase (Na(+)-K(+) pump). We explored the impact of acute and short-term training exercise on PLM physiology in human skeletal muscle. A group of moderately trained males (n = 8) performed a 1-h acute bout of exercise by utilizing a one-legged cycling protocol. Muscle biopsies were taken from vastus lateralis at 0 and 63 min (non-exercised leg) and 30 and 60 min (exercised leg). In a group of sedentary males (n = 9), we determined the effect of a 10-day intense aerobic cycle training on Na(+)-K(+)-ATPase subunit expression, PLM phosphorylation, and total PLM expression as well as PLM phosphorylation in response to acute exercise (1 h at ∼72% Vo(2peak)). Biopsies were taken at rest, immediately following, and 3 h after an acute exercise bout before and at the conclusion of the 10-day training study. PLM phosphorylation was increased both at Ser(63) and Ser(68) immediately after acute exercise (75%, P < 0.05, and 30%, P < 0.05, respectively). Short-term training had no adaptive effect on PLM phosphorylation at Ser(63) and Ser(68), nor was the total amount of PLM altered posttraining. The protein expressions of α(1)-, α(2)-,and β(1)-subunits of Na(+)-K(+)-ATPase were increased after training (113%, P < 0.05, 49%, P < 0.05, and 27%, P < 0.05, respectively). Whereas an acute bout of exercise increased the phosphorylation of PKCα/βII on Thr(638/641) pre- and posttraining, phosphorylation of PKCζ/λ on Thr(403/410) was increased in response to acute exercise only after the 10-day training. In conclusion, we show that only acute exercise, and not short-term training, increases phosphorylation of PLM on Ser(63) and Ser(68), and data from one-legged cycling indicate that this effect of exercise on PLM phosphorylation is not due to systemic factors. Our results provide evidence that phosphorylation of PLM may play a role in the acute regulation of the Na(+)-K(+)-ATPase response to exercise.

Influence of chronic and acute spinal cord injury on skeletal muscle Na+-K+-ATPase and phospholemman expression in humans

Na(+)-K(+)-ATPase is an integral membrane protein crucial for the maintenance of ion homeostasis and skeletal muscle contractibility. Skeletal muscle Na(+)-K(+)-ATPase content displays remarkable plasticity in response to long-term increase in physiological demand, such as exercise training. However, the adaptations in Na(+)-K(+)-ATPase function in response to a suddenly decreased and/or habitually low level of physical activity, especially after a spinal cord injury (SCI), are incompletely known. We tested the hypothesis that skeletal muscle content of Na(+)-K(+)-ATPase and the associated regulatory proteins from the FXYD family is altered in SCI patients in a manner dependent on the severity of the spinal cord lesion and postinjury level of physical activity. Three different groups were studied: 1) six subjects with chronic complete cervical SCI, 2) seven subjects with acute, complete cervical SCI, and 3) six subjects with acute, incomplete cervical SCI. The individuals in groups 2 and 3 were studied at months 1, 3, and 12 postinjury, whereas individuals with chronic SCI were compared with an able-bodied control group. Chronic complete SCI was associated with a marked decrease in [(3)H]ouabain binding site concentration in skeletal muscle as well as reduced protein content of the α(1)-, α(2)-, and β(1)-subunit of the Na(+)-K(+)-ATPase. In line with this finding, expression of the Na(+)-K(+)-ATPase α(1)- and α(2)-subunits progressively decreased during the first year after complete but not after incomplete SCI. The expression of the regulatory protein phospholemman (PLM or FXYD1) was attenuated after complete, but not incomplete, cervical SCI. In contrast, FXYD5 was substantially upregulated in patients with complete SCI. In conclusion, the severity of the spinal cord lesion and the level of postinjury physical activity in patients with SCI are important factors controlling the expression of Na(+)-K(+)-ATPase and its regulatory proteins PLM and FXYD5.

Differential expression of metabolic genes essential for glucose and lipid metabolism in skeletal muscle from spinal cord injured subjects

Skeletal muscle plays an important role in the regulation of energy homeostasis; therefore, the ability of skeletal muscle to adapt and alter metabolic gene expression in response to changes in physiological demands is critical for energy balance. Individuals with cervical spinal cord lesions are characterized by tetraplegia, impaired thermoregulation, and altered skeletal muscle morphology. We characterized skeletal muscle metabolic gene expression patterns, as well as protein content, in these individuals to assess the impact of spinal cord injury on critical determinants of skeletal muscle metabolism. Our results demonstrate that mRNA levels and protein expression of skeletal muscle genes essential for glucose storage are reduced, whereas expression of glycolytic genes is reciprocally increased in individuals with spinal cord injury. Furthermore, expression of genes essential for lipid oxidation is coordinately reduced in spinal cord injured subjects, consistent with a marked reduction of mitochondrial proteins. Thus spinal cord injury resulted in a profound and tightly coordinated change in skeletal muscle metabolic gene expression program that is associated with the aberrant metabolic features of the tissue.

Altered content of AMP-activated protein kinase isoforms in skeletal muscle from spinal cord injured subjects.

AMP-activated protein kinase (AMPK) is a pivotal regulator of energy homeostasis. Although downstream targets of AMPK are widely characterized, the physiological factors governing isoform expression of this protein kinase are largely unknown. Nerve/contractile activity has a major impact on the metabolic phenotype of skeletal muscle, therefore likely to influence AMPK isoform expression. Spinal cord injury represents an extreme form of physical inactivity, with concomitant changes in skeletal muscle metabolism. We assessed the influence of longstanding and recent spinal cord injury on protein abundance of AMPK isoforms in human skeletal muscle. We also determined muscle fiber type as a marker of glycolytic or oxidative metabolism. In subjects with longstanding complete injury, protein abundance of the AMPKγ3 subunit, as well as myosin heavy chain (MHC) IIa and IIx, were increased, whereas abundance of the AMPKγ1 subunit and MHC I were decreased. Similarly, abundance of AMPKγ3 and MHC IIa proteins were increased, whereas AMPKα2, -β1, and -γ1 subunits and MHC I abundance was decreased during the first year following injury, reflecting a more glycolytic phenotype of the skeletal muscle. However, in incomplete cervical lesions, partial recovery of muscle function attenuated the changes in the isoform profile of AMPK and MHC. Furthermore, exercise training (electrically stimulated leg cycling) partly normalized mRNA expression of AMPK isoforms. Thus, physical activity affects the relative expression of AMPK isoforms. In conclusion, skeletal muscle abundance of AMPK isoforms is related to physical activity and/or muscle fiber type. Thus, physical/neuromuscular activity is an important determinant of isoform abundance of AMPK and MCH. This further underscores the need for physical activity as part of a treatment regimen after spinal cord injury to maintain skeletal muscle metabolism.

MicroRNA‐208b progressively declines after spinal cord injury in humans and is inversely related to myostatin expression

The effects of long‐term physical inactivity on the expression of microRNAs involved in the regulation of skeletal muscle mass in humans are largely unknown. MicroRNAs are short, noncoding RNAs that fine‐tune target expression through mRNA degradation or by inhibiting protein translation. Intronic to the slow, type I, muscle fiber type genes MYH7 and MYH7b, microRNA‐208b and microRNA‐499‐5p are thought to fine‐tune the expression of genes important for muscle growth, such as myostatin. Spinal cord injured humans are characterized by both skeletal muscle atrophy and transformation toward fast‐twitch, type II fibers. We determined the expression of microRNA‐208b, microRNA‐499‐5p, and myostatin in human skeletal muscle after complete cervical spinal cord injury. We also determined whether these microRNAs altered myostatin expression in rodent skeletal muscle. A progressive decline in skeletal muscle microRNA‐208b and microRNA‐499‐5p expression occurred in humans during the first year after spinal cord injury and with long‐standing spinal cord injury. Expression of myostatin was inversely correlated with microRNA‐208b and microRNA‐499‐5p in human skeletal muscle after spinal cord injury. Overexpression of microRNA‐208b in intact mouse skeletal muscle decreased myostatin expression, whereas microRNA‐499‐5p was without effect. In conclusion, we provide evidence for an inverse relationship between expression of microRNA‐208b and its previously validated target myostatin in humans with severe skeletal muscle atrophy. Moreover, we provide direct evidence that microRNA‐208b overexpression decreases myostatin gene expression in intact rodent muscle. Our results implicate that microRNA‐208b modulates myostatin expression and this may play a role in the regulation of skeletal muscle mass following spinal cord injury.

Depressed expression of angiogenic growth factors in the subacute phase of myocardial ischemia: a mechanism behind the remodeling plateau?

Background and aimsTo investigate whether, in the subacute phase of acute myocardial infarction, in the peri-infarcted area the expressions of the vascular endothelial growth factor (VEGF-A) and angiopoietin (Ang) ligand receptors are depressed, and whether overexpression of these angiogens counteracts a downregulation of myocardial function. MethodsAcute myocardial infarction was induced by left anterior descending artery ligation and overexpression through injection of human VEGF-A165 and Ang-1 plasmids. The capillary and arteriolar densities, Akt-1 phosphorylation and citrate synthase activity were measured concurrent with the expression of VEGF-A, VEGFR1 and R2, Ang-1, Ang-2 and Tie-2. ResultsOne day after AMI, VEGR-2 was unchanged but all other measured factors in the two families were upregulated. After day 2, the Ang-2 expression increased but other measured factors decreased. After gene transfer, the vascular supply, Akt phosphorylation and citrate synthase activity were higher in the peri-infarcted area, where also the endogenous angiogenic growth factor expressions were increased. ConclusionA rapid decrease in angiogenic stimulating factors occurs in the subacute phase of AMI and is related to a progressive decrease in myocardial contraction. A negative consequence of such a circuit is a successive reduction in the vascular supply and contractility in areas with reduced perfusion. These negative adaptations can be counteracted by angiogen overexpression.